1. Field of the Invention
This invention aims to provide a method for the production of a novel fluorescence-labeled Fab'. It, therefore, relates to the fields of determination, analysis, and clinical diagnosis.
2. Prior Art Statement
Antibodies labeled with fluorescent substances are used for detection of biologically active molecules in various vital tissues and for detection of antigens present in very low concentrations in body fluids.
For the production of fluorescence-labeled antibodies, fluorescent substances such as fluorescein isocyanate and derivatives thereof have been widely used. However, the fluorescence-labeled antibodies produced using these substances have decreased the activity of the antibodies because these substances are conjugated to the antibodies by the NH.sub.2 group of the antibodies, which is the same part by which the antibodies bind to antigens.
As cases in which fluorescent substances reactive with the SH group have been used, there can be mentioned, for example, the production of fluorescence-labeled antibodies by the use of N-(7-dimethyl-4-amino-methylcumarinyl)maleimide disclosed in Japanese Published Unexamined Patent Application No. 55861/83 and that by the use of N-(3-pyrene)maleimide introduced by Robert P. Liburdy in Journal of Immunological Method, 28, (1979) pp. 233-242, for example. So far there has been no report relating to conjugation to the SH group of Fab'.
Similarly in the production of enzyme-labeled antibodies, enzymes are conjugated to antibodies through the medium of the NH.sub.2 group of the antibodies by the glutaraldehyde method or the periodate method, for example. Again in this case, it is known that these methods of enzyme-labeled antibodies are apt to decrease the activity of the antibodies and induce non-specific reactions. Ishikawa et al. have solved this problem by a method which conjugates an enzyme to the SH group of a given antibody through the medium of a maleimide compound possessing a maleimide group, namely a method which conjugates the enzyme to a different part from that at which the antibody binds to an antigen, specifically to the hinge region of the antibody.
Antibodies have five types, which are IgG, IgA, IgM, IgD, and IgE. Among these, IgG can be easily separated from antiserum and collected in high yields. IgG is composed of two heavy chains (H chains) and two light chains (L chains) and has the shape of the letter Y. Each H chain is coupled to each L chain by a disulfide bond (-S--S-) and the two H chains are also coupled to each other by a disulfide bond. The disulfide bonds of an antibody are severed through reduction with a reducing agent, into two SH groups. When the disulfide bond between an H chain and an L chain is reduced, the site at which the antibody is bound to an antigen is split at the same time. To avoid this trouble, the disulfide bond between the two H chains must be selectively reduced. Selective conversion of the disulfide bond between the two H chains to SH groups can be attained by digesting a given antibody with pepsin thereby producing F(ab').sub.2 and subsequently reducing the F(ab').sub.2 under relatively mild conditions. Fab' is obtained by reducing F(ab').sub.2. The hinge region is where the disulfide bond between the two H chains to be reduced is located.
The maleimide group of a maleimide compound tends to conjugate itself to the SH group and, therefore, is conjugated easily to the SH group of the Fab'. The method proposed by Ishikawa et al. attains the enzyme-labeled Fab' by the maleimide method and, therefore, does not impair the antigen binding activity.
Peroxidase, a labeling enzyme generally used in the conjugation of an enzyme with the Fab' has a molecular weight of 40,000, and the Fab' has a molecular weight of 47,000. Thus, they have nearly equal molecular weights. The enzyme-labeled Fab', therefore, has a molecular weight about twice that of peroxidase or Fab'. Separation of the unconjugated Fab' from the reacted product is easily attained by gel filtration (molecular sieve) by virtue of the difference in molecular weight.
Some fluorescent substances react with the SH group. It is, therefore, easily inferred that the production of a fluorescence-labeled Fab' will be attained by causing such a fluorescent substance to be conjugated to the SH group of the Fab'. Since the molecular weight of such a fluorescent substance is about 1,000 at most, it has been difficult to separate the fluorescence-labeled Fab' (molecular weight about 48,000) and the unconjugated Fab' (molecular weight about 47,000) by virtue of the difference of molecular weight. All attempts to obtain a purified fluorescence-labeled Fab' product from reaction of the SH group of the Fab' have failed.
Therefore, only fluorescence-labeled Fab' including unconjugated Fab' have been obtainable. Their F/P values (the number of molecules of the fluorescent substance per molecule of the antibody) have been low and their fluorescence intensities have been weak.